Method for ex vivo immunization using heterologous intact bispecific and/or trispecific antibodies

ABSTRACT

According to the invention there is described a method for ex vivo immunization of humans and animals comprising the following steps of:
         a) isolating autologous tumor cells;   b) treating the tumor cells to prevent the survival thereof following reinfusion;   c) incubating the thus treated tumor cells with intact heterologous bispecific and/or trisepcific antibodies showing the following properties:
           α—binding to a T cell;   β—binding to at least one antigen on a tumor cell;   γ—binding, by their Fc portion (in the case of bispecific antibodies), or by a third specificity (in the case of trispecific antibodies) to Fc receptor-positive cells.

This application is a continuation of U.S. patent application Ser. No.09/094,921, filed Jun. 15, 1998, now U.S. Pat. No. 7,018,632, whichclaims priority to German Patent Application No. 19725586.8, filed Jun.17, 1997.

The invention relates to a method for ex vivo immunization usingheterologous, intact bispecific and/or trispecific antibodies as well asthe use of the products of said method in the prevention and therapy oftumorous diseases and in particular in the induction of an anti-tumorimmunity.

Despite the progresses in chemotherapy and radiotherapy achieved duringrecent years, malignant diseases in humans, for example advanced breastcancer, still have an extraordinarily unfavorable prognosis. Generally,such diseases are impossible to heal. Therefore, it is necessary todevelop novel treatment strategies. In this respect, great hopes areplaced on immunotherapeutic approaches which shall be used to induce thepatient's immune system to reject the tumor. It is well known thattumor-associated antigens are present on tumor cells, and that inprinciple the immune system is very well able to recognize theseantigens and to attack the malignant cells. However, tumors havedeveloped various strategies which enable them to escape the immunereaction. They achieve this for example by an insufficient presentationof tumor-associated antigens and/or insufficient activation oftumor-specific T cells which are generally present.

With about 43,000 new cases/year, breast cancer occupies a top positionin the cancer statistics of women in Germany. Less than one third of thewomen suffering from lymph node invasion at the time of diagnosissurvive for 10 years without relapse.

To date, the immunotherapeutic approaches towards mamma carcinoma havebeen restricted to methods for unspecific stimulation, such as treatmentby BCG or levamisole, and to the use of LAK and NK cells with IL-2 (3,4). However, the types of immunotherapy employed provided no evidencefor a prolongation of life; the treatment by BCG even proved to bedisadvantageous (3). Since the unspecific activation of cells has notbeen very successful also in other types of tumor, attempts were madetowards the induction of a specific immune reaction.

For example, T cell-redirecting bispecific antibodies were used in tumortherapy. These antibodies bind with one of their binding arms to a Tcell receptor complex and with their other binding arm to atumor-associated antigen on a tumor cell. The resulting activation ofthe T cell and the spatial proximity of the tumor cell leads todestruction of the latter by induction of apoptosis or by cytokines,such as TNF-α or perforin, respectively.

The antibodies used in tumor therapy in the prior art were directlyinfused into patients. This type of procedure shows severaldisadvantages:

it requires high doses of antibodies;

severe side effects may occur;

by their tumor binding arm the antibodies may also bind to normal tissueduring in vivo application.

It is an object of the present invention to provide a novel method forthe therapy of malignant diseases in humans, in particular with theobjective of achieving an anti-tumor immunity.

According to the invention, this object has been achieved by the methodcharacterized in more detail in the present application. Preferredembodiments of the method are also provided herein.

The end product of the method of the present invention is a tumor cellpreparation containing antibodies. This tumor cell preparation is usedin the prevention and treatment of tumorous diseases by inducing ananti-tumor immunity.

By using the method of the present invention autologous tumor cells aretreated with heterologous bispecific and/or trispecific antibodies, andthe tumor cell preparation obtained by the present method is used forreinfusion into the patient or the animals from whom the autologoustumor cells have been obtained.

The invention relates further to the use of the method and the tumorcell preparations provided according to the invention in the preventionand therapy of tumorous diseases, in particular in the achievement of ananti-tumor immunity and particularly preferred of a long-term immunity.

The experiments provided in the present invention, particularly example2, show that a long-lasting tumor immunity is provided. The results ofthe experiment performed in mice can be transferred also to humans. Itis expected that a long-term immunity of several years can be providedby using the present invention. A tumor cell as described in the presentinvention is every cell which has lost its normal function by one ormore mutations or wherein its normal function has been changed. Due tothese mutations the tumor cells are able to propagate in an uncontrolledmanner.

Tumor immunity according to the present invention is defined byactivating the immune system of the body in an organism against theautologous tumor in such a way that a long-term or even permanentdestruction and/or control of the autologous tumor is achieved.

According to the invention every kind of tumors falling under thedefinition given above can be treated by the present method.Particularly epithelial tumors, adenocarcinomas, colon carcinomas, mammamammary carcinomas, overian carcinomas, carcinomas of lungs, throat,nose and ear can be treated. Furthermore, preferably non-epithelialtumors like leukemias and lymphomas and virus inducted tumors like livertumors or cervical carcinomas can be treated.

According to the invention, heterologous intact bispecific and/ortrispecific antibodies are used. These antibodies are contacted ex vivowith tumor cells (autologous tumor cells) previously obtained from apatient. To prevent the survival of tumor cells following reinfusion,the tumor cells were treated in a manner known per se, such as byirradiation, prior to contacting with the antibodies. Followingirradiation, the tumor cells are incubated with the intact heterologousbispecific and/or trispecific antibodies. According to the invention,not any antibody may be used but only antibodies which are intact, i.e.having a functional Fc portion, and they must be heterologous in nature,i.e. such antibodies which consist of heavy immunoglobulin chains ofdifferent subclasses (subclass combinations, also fragments) and/ororigin (species).

These intact heterologous bispecific and/or trispecific antibodies willbe selected to further have the following properties:

-   -   α—binding to a T cell;    -   β—binding to at least one antigen on a tumour cell;    -   γ—binding, by their Fc portion (in the case of bispecific        antibodies), or by a third specificity (in the case of        trispecific antibodies) to Fc receptor-positive cells.        In a particularly preferred embodiment of the present invention        the intact heterologous bispecific and/or trispecific antibodies        are selected to be able to activate the Fc receptor-positive        cell, and thereby inducing or increasing the expression of        cytokines and/or co-stimulatory antigens. The tumor cell        preparation obtained including said antibodies is them prepared        further for reinfusion. It is transferred for instance in a        device suitable for reinfusion.

In the case of trispecific antibodies, binding to the Fcreceptor-positive cells preferably takes place via the Fc receptor of Fcreceptor-positive cells or also via other antigens on Fcreceptor-positive cells (antigen-presenting cells), such as the mannosereceptor.

Only the present method and the use of the antibodies described hereinensures the development of an anti-tumor immunity after reinfusion ofthe antibodies into the patient from whom the tumor cells havepreviously been obtained. Preferably, reinfusion is carried out in apatient after treatment of the primary tumor, preferably in patients ina minimal residual disease (MRD) situation. In patients with fewresidual tumor cells but with a high risk of relapse, use of the methodprovided according to the invention will be particularly successful.

By using the method of the invention, it is possible to avoid thedisadvantages known from the prior art and described in more detailabove.

The heterologous bispecific and/or trispecific antibodies usefulaccording to the invention are in part known per se, but in part theyare described for the first time in the present application. An examplefor a bsab is antibody anti-CD3x anti-epcam which is employed inepithelial tumors such as mammary carcinoma.

According to the invention, two variations of the method may bedistinguished:

1. short-term incubation, and

2. long-term incubation.

A short-term incubation is an incubation of the autologous tumor cellswith intact heterologous bispecific and/or trispecific antibodies for aperiod of 10 minutes to 5 hours, or 10 minutes to 3 hours, or furtherpreferred for a period of about 15 minutes to 2 hours, further preferredfor a period of 15 minutes to 1 hour. The tumor cells charged withantibodies in this way are then prepared for reinfusion.

The long-term incubation is an incubation of the autologous tumor ellsalso for a period of about 10 minutes to 5 hours, preferably for aperiod of 15 minutes to 2 hours and further preferred for a period of 15minutes to 1 hour, so that the autologous tumor cells are charged withantibodies. Subsequently, blood cells of the patient, preferablymononucleated cells of the peripheral blood (PBMCs=peripheral bloodmononucleated cells) are added, and this mixture is then incubated overa prolonged period, such as 1 to 14 days, preferably 3 to 10 days andfurther preferred 6 to 10 days. Alternatively, another way of proceedingis contacting the autologous tumor cells directly with the bispecificand/or trispecific antibodies and with the patient's blood cells,preferably peripheral blood mononucleated cells. In this way, “priming”of numerous immune cells against the tumor is achieved already ex vivo.Afterwards, these cells are reinfused into the patient. Long-termincubation also leads to internalization and degradation of theantibodies.

Preliminary in vitro results show that immune cells pre-treated in theway described are able to destroy tumor cells without further additionof bispecific and/or trispecific antibodies (cf. Example 1).

In short-term as well as long-term incubation, the T cells areredirected to the tumor ells by the bispecific and/or trispecificantibodies which are immobilized on the tumor cells; at the same timebinding of Fc receptor-positive cells to the Fc portion of thebispecific and/or trispecific antibody takes place after reinfusion.This leads to activation of Fc receptor-positive cells by their bindingto the Fc portions of immobilized (on the T cell or tumor cell,respectively) intact bispecific antibodies.

To enhance the success of immunization, the tumor cells treated with theantibodies either according to the short-term incubation method or thelong-term incubation method may be administered to the patient not onlyonce but optionally also several times.

On the tumor cell, an up-regulation of MHC 1 as well as activation ofthe intracellular processing machinery (proteasome complex) occurs dueto the release of cytokines (such as INF-γand TNF-α) in direct proximityof the tumor cell. Cytokines are released because of the bispecificantibody-mediated activation of T cells and accessory cells (see FIGS. 1and 3). I.e. by the intact bsab not only tumor cells are destroyed andphagocytized but indirectly also their anti-tumor immunity is increased.

Activation of the Fc receptor-positive cells by the bsab depends on thesubclass or subclass combination, respectively, of the bsab. Asdemonstrated in in vitro experiments, for example bsabs of the subclasscombination mouse IgG2a/rat IgG2b are able simultaneously to bind to andactivate Fc receptor-positive cells leading to up-regulation andformation (expression), respectively, of co-stimulatory antigens, suchas CD40, CD80, or CD86, on the cell surface of such cells. In contrast,bsabs of the subclass combination mouse IgG1/IgG2b are able to bind toFc receptor-positive cells (1) but clearly are unable to activate thesecells to a comparable extend (2).

While the intact bsab at the same time binds to and activates the T cellvia one of its binding arms (e.g. to CD3 or CD2), co-stimulatory signalsderived from the Fc receptor-positive cell bound to the Fc portion ofthe bsab may be transferred to the T cell. I.e. only the combination ofT cell activation via one binding arm of the bsab and the concomitanttransfer of co-stimulatory signals from the Fc receptor-positive cell tothe T cell results in an effective T cell activation (FIG. 1A). Tumorspecific T cells which have been insufficiently activated at the tumorcell and are anergic may also be reactivated according to the ex vivopre-treatment of the invention (FIG. 1B).

A further important aspect in the induction of anti-tumor immunity isthe possibility of phagocytosis, processing and presentation of tumorcomponents by accessory cells (monocytes/macrophages, dendritic cells,and NK—“natural killer”—cells) which have been directed and activated bythe bsab. By this classical mechanism of antigen presentationtumor-specific CD4 cells as well as CD8 positive cells can be generated.Moreover, tumor-specific CD4 cells play an important role in theinduction of a humoral immune reaction in the context of the T-B cellcooperation.

Bispecific and trispecific antibodies are able to bind to the T cellreceptor complex of the T cell by one binding arm and totumor-associated antigens on the tumor cells by the second binding arm.Thereby, they activate T cells which destroy the tumor cells byreleasing cytokines or apoptosis-mediating mechanisms. Furthermore, inthe context of their activation by bispecific antibodies it is clearlypossible for T cells to recognize tumor-specific antigens via theirreceptor whereby a long-lasting immunization is initiated (FIG. 1B). Inthis respect, the intact Fc portion of the bispecific or trispecificantibody is of particular importance mediating the binding to accessorycells such as monocytes/-macrophages and dendritic cells and inducingthese cells to become themselves cytotoxic and/or simultaneouslytransfer important co-stimulatory signals to the T cell (FIG. 1B). Inthis manner, it seems to be possible that a T cell reaction may beinduced also against so far unknown tumor-specific peptides.

Redirection of possibly anergized tumour-specific T cells to tumor cellsby means of bispecific and/or trispecific antibodies and concomitantco-stimulation of such T cells by accessory cells bound to the Fcportion of the bispecific or trispecific antibody might act to reversethe anergy of cytotoxic T cells (CTLs). I.e. using intact heterologousbispecific and/or trispecific antibodies a T cell tolerance existing inthe patient against the tumor may be neutralized and, thereby, along-lasting anti-tumor immunity may be induced.

The last aspect is supported by preliminary in vivo data fromexperiments with mice indicating a long-lasting anti-tumor immunityfollowing treatment with a syngeneic tumor and intact bsab. In theseexperiments a total of 14 out of 14 animals which could be successfullytreated with bsab after a first tumor injection survived another tumorinjection 144 days after the first one—without further administration ofbsab (see Example 2).

Further advantages in the ex vivo immunization by bispecific and/ortrispecific antibodies are (i) minimizing possible side effects, (ii)controlled binding of the tumor binding arm to the tumor cells outsideof the body, and (iii) use of as little bispecific and trispecificantibodies as possible. Principally, there are two different ways ofproceeding which will be detailed in the following. An important aspectwith long-term incubation is that the bispecific or trispecific antibodyemployed is exhausted and degraded during the incubation period planned.In this way, this immunization would avoid the lengthy drug approvalprocess.

In the short-term and long-term incubation procedures, the tumor cellsare incubated with antibodies over a period of 10 minutes to 5 hours,preferably up to 3 hours, further preferred up to 2 hours and stillfurther preferred 15 minutes to 1 hour. Preferably, the incubation iscarried out at a temperature of 4° C. to 25° C., particularly preferred4° C. to 10° C. The incubation is preferably performed in a sterileenvironment in bufferd saline having a neutral pH. In the case ofshort-term incubation, reinfusion into the patient is performedimmediately afterwards. In the long-term incubation procedure, followingthis preincubation mononucleated peripheral blood cells are added andincubated together with the preincubated tumor cells/antibodies for afurther period of 1 to 14 days, more preferably 3 to 10 days, furtherpreferred 6 to 10 days. Preferably, this incubation is performed at 37°C. under sterile conditions as well as under GMP conditions (GoodManufacturing Production=GMP) in an incubator. As detailed above, inlong-term incubation the blood cells may alternatively be incubatedtogether with tumor cells and antibodies under suitable conditions.

The incubation conditions described above are only intended to be anexample. Depending on the tumor cells and the antibodies used also othertime periods, temperature conditions etc., and in general differentincubation conditions may be used. By simple experimentation, theskilled artisan will be able to establish such conditions.

During preincubation the tumor cells are preferably employed in anamount of 10⁷ to 10⁹ cells, further preferred in an amount of about 10⁸cells. The peripheral blood mononucleated cells are added in an amountof about 10⁸ to 10¹⁰ cells. Naturally, the skilled artisan may selectdifferent incubation conditions which may be determined by laboratoryexperimentation (for example changes in cell number and incubationperiod). The bi-specific and/or tri-specific antibodies used in themethod of the present invention are added in an amount of 2 to 100 μg,more preferably 5 to 70 μg, particularly preferred 5 to 50 μg.

The autologous tumor cells employed are for example irradiated toprevent further survival of tumor cells. For example, gamma radiation isused e.g. employed in a radiation dose of 50 to 100 Gy. In anotherembodiment of the present invention the autologous tumor cells aretreated by chemical substances, for instance by mitomycin C to preventtheir further survival.

The antibodies used according to the invention are preferably able toreactivate tumor-specific T cells being in an anergic state. Further,they are able to induce tumor-reactive complement-binding antibodies andthereby a humoral immune reaction.

Binding preferably takes place via CD3, CD2, CD4, CD5, CD6, CD8, CD28,and/or CD44 to the T cell. Fc receptor-positive cells at least bear aFcγ receptor I, II, or III.

Antibodies which may be employed according to the invention are able tobind to monocytes, macrophages, dendritic cells, “natural killer” cells(NK cells) and/or activated neutrophils being Fcγ receptor 1-positivecells.

The antibodies which may be used according to the invention lead to aninduction or increase in the expression of CD40, CD80, CD86, ICAM-1,and/or LFA-3 as co-stimulatory antigens and/or cytokine secretion by theFc receptor-positive cell. The cytokines preferably are IL-1, IL-2,IL-4, IL-6, IL-8, IL-12, and/or TNF-α.

Binding to the T cell preferably takes place via the T cell receptorcomplex of the T cell.

The bispecific antibodies which may be used according to the inventionpreferably are:

-   -   an anti-CD3 X anti-tumor-associated antigen antibody and/or        anti-CD4 X anti-tumor-associated antigen antibody and/or        anti-CD5 X anti-tumor-associated antigen antibody and/or        anti-CD6 X anti-tumor-associated antigen antibody and/or        anti-CD8 X anti-tumor-associated antigen antibody and/or        anti-CD2 X anti-tumor-associated antigen antibody and/or        anti-CD28 X anti-tumour-associated antigen antibody and/or        anti-CD44 X anti-tumor-associated antigen antibody.

The trispecific antibodies which may be employed according to theinvention preferably are:

-   -   an anti-CD3 X anti-tumor-associated antigen antibody and/or        anti-CD4 X anti-tumor-associated antigen antibody and/or        anti-CD5 X anti-tumor-associated antigen antibody and/or        anti-CD6 X anti-tumor-associated antigen antibody and/or        anti-CD8 X anti-tumor-associated antigen antibody and/or        anti-CD2 X anti-tumor-associated antigen antibody and/or        anti-CD28 X anti-tumor-associated antigen antibody and/or        anti-CD44 X anti-tumor-associated antigen antibody.

The trispecific antibodies useful according to the invention at leasthave a T cell binding arm, a tumor cell binding arm and a binding armwhich binds to Fc receptor positive cells. The latter of the bindingarms mentioned may be an anti-Fc receptor binding arm or a mannosereceptor binding arm.

The bispecific antibody preferably is a heterologous intact rat/mousebispecific antibody.

By the bispecific and trispecific antibodies useful according to theinvention T cells are activated and redirected against the tumor cells.Heterologous intact bispecific antibodies which may be preferably usedare selected from one or more of the following isotype combinations:

-   -   rat-IgG2b/mouse-IgG2a,    -   rat-IgG2b/mouse-IgG2b,    -   rat-IgG2b/mouse-IgG3;    -   rat-IgG2b/human-IgG1,    -   rat-IgG2b/human-IgG2,    -   rat-IgG2b/human-IgG3[oriental allotype G3m(st)=binding to        protein A],    -   rat-IgG2b/human-IgG4;    -   rat-IgG2b/rat-IgG2c;    -   mouse-IgG2a/human-IgG3[caucasian allotypes G3m(b+g)=no binding        to protein A, in the following indicated as *]    -   mouse-IgG2a/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]    -   mouse-IgG2a/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]    -   mouse-IgG2a/human-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]    -   mouse-[VH-CH1,VL-CL]-human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]    -   mouse-[VH-CH1,VL-CL]-human-IgG4/rat-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4[N-terminal        region of CH2]-human-IgG3*[C-terminal region of CH2: >as        position 251]-human-IgG3*[CH3]    -   rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge-CH2-CH3]    -   rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG2-[hinge-CH2-CH3]    -   rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG3-[hinge-CH2-CH3,        oriental allotype]    -   rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG4-[hinge-CH2-CH3]    -   human-IgG1/human-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]    -   human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4[N-terminal        region of CH2]-human-IgG3*[C-terminal region of CH2: >as        position 251]-human-IgG3*[CH3]    -   human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4[N-terminal        region of CH2]-human-IgG3*[C-terminal region of CH2: >aa        position 251]-human-IgG3*[CH3]        human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG2[N-terminal        region of CH2]-human-IgG3*[C-terminal region of CH2: >aa        position 251]-human-IgG3*[CH3]    -   human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG2        [N-terminal region of CH2]-human-IgG3*[C-terminal region of        CH2: >aa position 251]-human-IgG3*[CH3]    -   human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]    -   human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]    -   human-IgG2/human-[VH-CH1,VL-CL]-human-IgG2-[hinge]-human-IgG3*-[CH2-CH3]    -   human-IgG4/human-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG3*-[CH2-CH3]    -   human-IgG4/human-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4[N-terminal        region of CH2]-human-IgG3*[C-terminal region of CH2: >aa        position 251]-human-IgG3*[CH3]        mouse-IgG2b/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]    -   mouse-IgG2b/human-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]    -   mouse-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]    -   mouse-[VH-CH1,VL-CL]-human-IgG4/rat-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4-[CH2]-human-IgG3*-[CH3]    -   human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4-[CH2]-human-IgG3*-[CH3]    -   human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4-[CH2]-human-IgG3*-[CH3]    -   human-IgG4/human-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4-[CH2]-human-IgG3*-[CH3]

The antibodies useful according to the invention preferably aremonoclonal, chimeric, recombinant, synthetic, semi-synthetic orchemically modified intact antibodies having for example Fv, Fab, scFvor F(ab)₂ fragments.

Preferably used are antibodies or derivatives or fragments of humanorigin or antibodies altered in a way that makes them suitable forapplication to humans (so-called “humanized antibodies”) (see forexample Shalaby et al., J. Exp. Med. 175 (1992), 217; Mocikat et al.,Transplantation 57 (1994), 405).

The preparation of the various types of antibodies and fragmentsmentioned above is obvious to one skilled in the art. The preparation ofmonoclonal antibodies preferably originating from mammals, e.g. humans,rat, mouse, rabbit or goat, may be performed using conventional methods,as for example described in Köhler and Milstein (Nature 256 (1975),495), in Harlow and Lane (Antibodies, A Laboratory Manual (1988), ColdSpring Harbour) or in Galfré (Meth. Enzymol. 73 (1981), 3).

Furthermore, it is possible to prepare the antibodies described by meansof recombinant DNA technology according to techniques obvious to theskilled artisan (see Kurucz et al., J. Immunol. 154 (1995), 4576;Hollinger et al., Proc. Natl. Acad. Sc. USA 90 (1993), 6444).

The antibodies used in the present method can be designed andmanufactured by a person skilled in the art without undue burden. Theenclosed list of references, particularly references (7) to (11)describe methods on how to obtain bispecific and trispecific antibodiesto be used in the present invention.

Particularly document (9) of Greenwood et al. discloses the exchange ofsingle immunoglobulin domains (for instance CH2) by suitable cloningtechnique. By using these cloning techniques novel antibody combinationsdescribed herein can be provided. Examples are:

human-(VH-CH1, VL-CL)-human IgG4-(hinge)-human IgG4 (N-terminale regionof CH2)-human IgG3*(C-terminal region of CH2:>aminoacid position251)-human IgG3*(CH3).

The combination with an antibody: human IgG4 for the preparation of thebispecific antibody: human IgG4/human-(VHCH1, VL-CL)-humanIgG4-(hinge)-human IgG4 (N-terminal region of CH2)-humanIgG3*(C-terminal region of CH2:>aminoacid position 251)-human IgG3*(CH3)is prepared by simple cell fusion as described for instance in document(6).

On the one hand, the preparation of antibodies having two differentspecificities, the so-called bispecific antibodies, may be performedusing recombinant DNA technology, but on the other hand also by theso-called hybrid-hydridoma fusion technique (see for example Milstein etal., Nature 305 (1983), 537). By this technique, hybridoma cell linesproducing antibodies each having one of the desired specificities arefused, and recombinant cell lines producing antibodies with bothspecificities are identified and isolated.

The problem underlying the invention may be solved both by bispecificand by trispecific antibodies insofar as they show the features andactivities characterized herein. In the following, the preparation ofantibodies having two and three specificities is described in moredetail. To provide such bispecific and trispecific antibodies belongs tothe state of the art, and the literature describing such methods ofpreparation is hereby incorporated by reference in its entirety.

The preparation of antibodies having three specificities, so-calledtrispecific antibodies, which are also suitable to solve the fundamentalproblem of the invention may be for example carried out by coupling toone of the heavy IgG chains of a bispecific antibody a thirdantigen-binding site having another specificity, e.g. in the form of“single chain variable fragments” (scFv). The scFv may be for examplebound to one of the heavy chains via a

-   -   -S-S(G₄S)_(n)D-I linker        (S=serine, G=glycine, D=aspartate, I=isoleucine).

Analogously, trispecific F(ab)₂ constructs may be prepared substitutingthe CH2-CH3 regions of the heavy chain of one specificity of abispecific antibody by a scFv of a third specificity while the CH2-CH3regions of the heavy chain of the other specificity are removed, e.g. byintroduction of a stop codon (at the end of the “hinge” region) into thecoding gene for example by homologous recombination (see FIG. 5).

It is also possible to prepare trispecific scFv constructs. In this casethree VH-VL regions representing three different specificities arearranged in series (FIG. 6).

According to the invention, there are for example used intact bispecificantibodies. Intact bispecific antibodies are a combination of twoantibody semi-molecules (each of one H and L immunoglobulin chain) eachrepresenting one specificity and, like normal antibodies, having inaddition a Fc portion which performs the well known effector functions.Preferably, they are prepared by quadroma technology. This method ofpreparation is described representatively in DE-A-44 19 399. Thisdocument is incorporated by reference in its entirety for the purpose ofcomplete disclosure also with respect to a definition of bispecificantibodies. Naturally, also other methods of preparation may be employedas long as they result in the intact bispecific antibodies defined aboverequired according to the invention.

For example, by a newly developed method of production (6) intactbispecific antibodies may be prepared in a sufficient amount. Thecombination of 2 bispecific antibodies against 2 differenttumor-associated antigens (e.g. c-erb-B2, ep-cam, such as GA-733-2=C215)on the mamma carcinoma cells minimizes the risk that tumor cellsexpressing only one antigen are not recognized.

There have also been attempts to achieve an anti-tumor immunity bytreatment with bispecific F(ab′)2 fragments having the specificities ofanti-c-erb-B2 x anti CD64. The main disadvantage of bsF(ab′)2 fragmentsis that due to the specificities used only FcγRI+ cells are redirectedto the tumor. T cells are not redirected to the tumor by this bispecificantibody. While bsF(ab′)2 fragments have the potential to directlydestroy the tumor, they are unable to establish an anti-tumor immunitythemselves. Only the T cell with its specific T cell receptor has thiscapability. While the FcγRI+ cells are able to indirectly activatetumor-specific T cells by presenting tumor specific peptides (via MHCIor MHCII, respectively), for example following phagocytosis of tumorcomponents, the efficiency of induction of an anti-tumor immunity isthis case is not as high (only in 30% of the patients).

Further advantages of intact bsabs capable of redirecting T cells ascompared to the above-mentioned bsF(ab′)2 fragments are detailed in thefollowing:

-   1. To intact bsabs there may bind Fc receptor-positive cells and may    on the one hand by ADCC (antibody-dependent cell-mediated    cytotoxicity) contribute directly to the destruction of the tumor    and on the other hand to T cell activation, as detailed above.-   2. By intact T cell-redirecting bsabs also anergize tumor-specific T    cells are directed to the tumor cell which according to the    invention may be directly reactivated at the tumor. This may not be    achieved using a bsF(ab′)2 fragment having the specificities of anti    CD64 x anti tumor-associated antigen.-   3. A bsF(ab′)2 fragment having the specificities of anti CD64 x anti    tumor-associated antigen is merely able of achieving an anti-tumor    immunity in 30% of the patients while according to the invention in    experiments with mice using T cell-redirecting intact bsabs a    protection in 100% of the animals could be achieved.

Binding of the bsabs to Fcγ-RI has two significant advantages withrespect to optimum anti-tumor effectivity:

-   (1) Fey-RI-positive cells are capable of eliminating tumor cells by    means of ADCC (11) and in this respect may contribute    synergistically to the anti-tumor-effect of the cytotoxic T cells    which have been directed to the tumor cell by the bsab (13).-   (2) Fcγ-RI-positive cells (such as monocytes/macrophages/dendrites)    are capable of providing important co-stimulatory signals similar to    antigen presentation to the T cell and thereby to prevent anergizing    of the T cell. Furthermore, as shown in FIG. 1, as a desired side    product due to the intact bsab-mediated interaction of the T cell    with accessory cell and tumor cell there may be stimulated T cells    having a T cell receptor which recognizes tumor-specific peptides    (presented on the tumor cell via MHC antigens). The co-stimuli    necessary for a correct activation of the T cell in this situation    would be provided by the accessory cell (e.g. the monocyte). In this    respect, the antibody of the invention besides the direct T cell    receptor-independent bsab-mediated tumor destruction (FIG. 1A)    should also activate and generate tumor-specific T cells (FIG. 1B)    which after degradation of the bsabs continue to patrol in the    patient. I.e. by means of intact bsabs similar to gene therapy    approaches (e.g. by incorporation of co-stimulatory antigens such as    B-7 into the tumor cell) the tumor tolerance in the patient may be    overcome.

In this respect, it is further beneficial that the expression of Fcγ-RIis up-regulated on the respective cells following G-CSF treatment.

The invention has been described in the above and will be described inthe following in particular with respect to bispecific antibodies.Instead of bispecific antibody, of course also trispecific antibodiesmay be used as long as they comply with the provisions made.

The invention has been and will be described with respect to theaccompanying Figures. The Figures show:

FIG. 1: the role of accessory cells in tumor immunotherapy by means ofbispecific antibodies;

FIG. 2: the destruction of tumor cells following administration ofbispecific antibodies as evidenced by flow-cytometry;

FIG. 3: induction of cytokines by intact bispecific antibodies only butnot by parental antibodies;

FIG. 4: efficiency of the method according to the invention in vivo;

FIG. 5: trispecific F(ab)₂ antibodies;

FIG. 6: trispecific scFv antibody.

IMMUNIZATION PROTOCOLS

Ex Vivo Immunization (Short-Term Incubation)

-   1. Preparation of a single cell suspension (10⁷-10⁹ cells) from    autologous tumor material (or allogenic tumor cells of the same    tumor type) with subsequent γ irradiation (50-100 Gy).-   2. Addition of bsabs (5-50 μg) and incubation for 45 minutes at    4° C. Afterwards washing away of unbound antibodies.-   3. Reinfusion of the cell mixture (i.v.).    Ex Vivo Immunization (Long-Term Incubation)-   1. Preparation of a single cell suspension (10⁷-10⁹ cells) from    autologous tumor material (or allogenic tumor cells of the same    “tumor type) with subsequent y irradiation (50-100 Gy).-   2. Addition of bsabs (5-50 μg), 45 minutes incubation.-   3. Addition of PBMCs (10⁸-10¹⁰), [alternatively: 1×10⁹ cells    obtained from T cell aphaeresis].-   4. After 5 to 7 days monitoring of T cell reactivity by transfer of    aliquots e.g. to allogenic breast cancer cell lines (MCF-7, MX-1).-   5. Reinfusion (i.v.) of the cultured PBMCs on days 4 to 14 into the    patient (in the case of T cell aphaeresis: cryo conservation).    Abbreviations: PBMCs, Peripheral Blood Mononucleated Cells; i.v.,    Intravenously.

A similar assay but instead depending on the addition of cytokines andcarried out using conventional bsabs (no activation of accessory cellsby bsabs of the subclass combination rat IgG2B x rat IgG1) demonstratesthe principal effectivity of such an ex vivo immunization in the animalmodel (5).

In contrast to this, the advantage of the method disclosed hereinresides in the “self-sufficiency” with respect to cytokines (such asINF-α or TNF-α) required for an up-regulation of for example MHC 1 onthe tumor cell by simultaneous activation of T cells and accessory cells(monocytes/macrophages, Fig.) on the tumor cell. This is achieved by theparticular subclass combination mentioned at the beginning of the intactbsab used herein. In the case of short-term incubation these processestake place in the patient. Further advantages in short-term incubationare (i) avoiding the cultivation of the cell suspension withserum-containing medium otherwise necessary. (ii) Due to this, also thecost-intensive cultivation according to GMP regulations may be omitted.(iii) A further important aspect is avoidance or reduction,respectively, of possible side effects by the bsab because of thesignificantly lower amount of antibodies applied.

An advantage in long-term incubation is that the bsab in vitro aftersome time exhausts itself (and, thus, this method may be established notas a medicament but as a “medical device”).

EXAMPLE 1 Bispecific Antibody-Mediated Lysis of Tumor Cells by AllogenicT Cells

H-Lac78 is a cell line which has been established from a hypopharynxcarcinoma and which expresses epcam to a high extent (own FACS data).Using H-Lac78 and peripheral mononucleated cells (PBMC) from volunteersit was possible to detect the generation of allogenic cytotoxic Tlymphocytes. For this purpose, constant amounts of H-Lac78 (2×10⁴) wereincubated with varying amounts of PBMCs in the presence (10 ng) orabsence of a bsab (anti epcam x anti CD3). After a period of seven daysthe PBMCs were removed and analyzed in a flow-cytometer. At the sametime, the number of H-Lac78 tumor cells was determined. The activationof T cells may be observed microscopically by means of clusterformation; proliferation may be evidenced by the incorporation ofradiolabeled thymidine. The detection of remaining tumor cells isperformed microscopically as well as by the epithelial marker epcamwhich is not expressed on peripheral blood cells. As shown in FIG. 2,the H-Lac78 cells were completely lysed in the presence of bsab, i.e. noepcam-positive cells were detectable in the flow-cytometer after sevendays. These data were confirmed by microscopic observations. Incontrast, without bsab a confluent layer of H-Lac78 cells was observedin the wells and epcam-positive cells were detectable by FACS.

Detection of Activated Allospecific CTLS by Transfer Experiment

In a subsequent transfer experiment the PBMCs incubated with or withoutbsab, respectively, were transferred onto new H-Lac78 cells withoutreaddition of bsab. Also in this case, the tumor cells were lysed butexclusively by PBMCs which had been activated by

-   bsab previously. H-Lac78 lysis was complete within 24 hours up to a    ratio of 2 PBMCs to 1H-Lac78 cell. This result indicates the    generation of allospecific CTLs without external addition of    interleukin-2 (IL-2). Since IL-2 is essential for the activation of    T lymphocytes, the data obtained herein suggest that by    bsab-mediated activation IL-2 is produced by the T cells themselves.    Induction of IL-2 mRNA by addition of bsab could be confirmed    afterwards by RT-PCR where the bsab was clearly superior to the    parental starting antibodies (FIG. 3). This observation is important    insofar as IL-2 has been described as an anti-tumor effective    cytokine; but the systemic administration of which in an appropriate    concentration is limited because of its toxicity. In contrast, the    risk of toxicity does not appear in the local production of IL-2 as    it is for example induced by intact bsab. Also, since an effective    induction of IL-2 (and IL-12) requires stimulation of T cells via    the T cell receptor and CD28, this indicates the importance of Fc    receptor-positive cells (providing the ligands for CD28, CD80, and    CD86) in T cell activation by intact bsab.

EXAMPLE 2

To address the question whether bispecific antibodies are able to inducea long-lasting anti-tumor immunity C57BL/6 mice were first injected with5×10³ syngeneic B16 tumor cells. Two days later, a group of mice (numberof 18) were treated with intact bsab prepared by quadroma technology (6)and recognizing a target structure (ep-cam/C215=tumor-associatedantigen) on the tumor cell as well as CD3 on the T cells. A second group(number of 6) received an equimolar amount of Fab fragments of both ofthe specificities contained in the bsab only. While all of the animalsof the Fab control group died or had to be sacrificed within 56 days, 14of the 18 animals treated with bsab survived. 144 days after the firstinjection of tumor cells the 14 surviving animals were injected withanother dose of 750 B16 tumor cells but this time without administrationof bsabs. As a control, the same number of tumor cells was administeredto 5 untreated animals. While the last animal of the untreated controlgroup had to be sacrificed 66 days after tumor injection, all of theanimals treated with bsab survived (monitoring period: 120 daysfollowing second tumor cell injection). See also FIGS. 4A and B:Survival graphs of the two subsequent experiments described above.

1. A composition comprising activated peripheral blood mononucleatedcells and non-viable tumor cells from the same individual, thecomposition obtained by incubating ex vivo: a) tumor cells isolated froma patient and treated to prevent survival following reinfusion into thepatient; b) peripheral blood mononucleated cells from the patient; andc) an intact heterologous bispecific antibody showing the followingproperties: (i) binding to a T cell; (ii) binding to at least onetumor-associated antigen on the tumor cells; (iii) binding, by their Fcportion to Fc receptor-positive cells; and (iv) activating the Fcreceptor-positive cells, thereby inducing or increasing the expressionof a cytokine and/or a co-stimulatory antigen, wherein the bispecificantibody has an isotype combination selected from the group consistingof: rat-IgG2b/human-IgG1, rat-IgG2b/human-IgG2,rat-IgG2b/human-IgG3[oriental allotype G3m(st)=binding to protein A],rat-IgG2b/human-IgG4, rat-IgG2b/rat-IgG2c,mouse-IgG2a/human-IgG3[caucasian allotypes G3m(b+g)=no binding toprotein A, in the following indicated as *], mouse-IgG2a/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],mouse-IgG2a/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],mouse-IgG2a/human-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],mouse-[VH-CH1,VL-CL]-human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],mouse-[VH-CH1,VL-CL]-human-IgG4/rat-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4[N-terminalregion of CH2]-human-IgG3*[C-terminal region of CH2: >aa position251]-human-IgG3*[CH3],rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge-CH2-CH3],rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG2-[hinge-CH2-CH3],rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG3-[hinge-CH2-CH3, orientalallotype], rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG4-[hinge-CH2-CH3],human-IgG1/human-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4[N-terminalregion of CH2]-human-IgG3*[C-terminal region of CH2: >aa position251]-human-IgG3*[CH3],human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4[N-terminalregion of CH2]-human-IgG3*[C-terminal region of CH2: >aa position251]-human-IgG3*[CH3], human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG2[N-terminal region ofCH2]-human-IgG3*[C-terminal region of CH2: >aa position251]-human-IgG3*[CH3],human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG2[N-terminalregion of CH2]-human-IgG3*[C-terminal region of CH2: >aa position251]-human-IgG3*[CH3],human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG-[hinge]-human-IgG3*-[CH2-CH3],human-IgG2/human-[VH-CH1,VL-CL]-human-IgG2-[hinge]-human-IgG3*-[CH2-CH3],human-IgG4/human-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG3*-[CH2-CH3],human-IgG4/human-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4[N-terminalregion of CH2]-human-IgG3*[C-terminal region of CH2: >aa position251]-human-IgG3*[CH3],mouse-IgG2b/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],mouse-IgG2b/human-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],mouse-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],mouse-[VH-CH1,VL-CL]-human-IgG4/rat-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4-[CH2]-human-IgG3*-[CH3],human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4-[CH2]-human-IgG3*-[CH3],human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4-[CH2]-human-IgG3*-[CH3],human-IgG4/human-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4-[CH2]-human-IgG3*-[CH3],rat-IgG2b/mouse-IgG2a, rat-IgG2b/mouse-IgG2b, and rat-IgG2b/mouse-IgG3.2. The composition of claim 1, which is incubated for 1-14 days.
 3. Thecomposition of claim 2, which is incubated for 3 to 10 days.
 4. Thecomposition of claim 3, which is incubated for 6 to 10 days.
 5. A methodfor inducing an anti-tumor immunity in a patient, comprising the step ofadministering to the patient the composition of any one of claims 1-4.6. The method of claim 5, wherein the administration step is performedmultiple times.